The broad range goals of the proposed research involves development of MALDI Imaging Mass Spectrometry (IMS) to achieve (i) next generation improvements of the technology and associated methodologies for both profiling and imaging of tissues to gain significant improvements in speed, sensitivity and image resolution, (ii) development, testing, and validation of in situ chemistries for targeted analysis of specific proteins or families of proteins at high sensitivity, including in situ protein identification and (iii) applications of IMS to several biological research problems that can significantly benefit from this technology, e.g., addressing several fundamental questions in neurobiology, reproductive biology (embryo implantation), and drug distribution, toxicity and efficacy in intact organs and animal sections. More specifically, work proposed for technology and methodology improvements involve next generation enhancements for image acquisition speed, image resolution, and ease, robustness, and reproducibility of image acquisition. New sample preparation protocols will be devised to facilitate histology-directed molecular tissue analysis and to provide ease of 3-D protein image construction of tissue structures and small organs. Targeted analysis of proteins in tissues using in situ chemistries (tissue-based protein arrays) will be developed for the identification and quantitation of proteins directly in tissue sections. These include protocols that allow protease digestions in micron sized areas in tissue and the use of submicron particles having light cleavable linkers attached to molecular probes to remove and analyze proteins or families of proteins at very high sensitivity with spatially fidelity to the original tissue. Finally, these technologies and protocols will be applied to specific research problems to establish their efficacy in helping solve biological problems and provide spatially resolved molecular information. These applications will include problems in neurobiology, including studies in a mouse model of Parkinsonism, the creation of 3-D maps of mouse brain proteins, studies of embryo implantation in mouse, and applications to drug analysis, metabolism, and efficacy in studies in rat and mouse through tissue, organ, and whole body analyses. This research will provide a new technology for the discovery of molecular patterns in both health and disease. The elucidation of the spatial orientation of proteins in tissues and their quantitation and specific identification will answer many scientific questions about how disease might form and spread and will bring new insights into the prevention and treatment of human disease and the maintenance of human health.